1. Field of the Invention
The present invention relates to a method for the validation of a non-particulate ion exchange adsorber and a kit for the validation of a non-particulate ion exchange adsorber.
2. Description of the Related Art
The present invention is based on the definitions described below. “Adsorptive substance separation” is understood to mean the separation of one or more components from a fluid phase by selective adsorption of this/these component(s) on a solid phase, the “adsorbent” (plural “adsorbents”). The field of the invention relates to substance separation in liquids, the liquid being called the “medium” below and the device in which the adsorption is performed the “adsorber”. Adsorbents are porous solids which via functional surface groups, which are called “ligands”, can selectively enter into bonds with certain components of fluids. As well as the long known “particulate” adsorbents, also called chromatography gels, other “non-particulate adsorbents” have become established, which are based on a matrix of an entirely different nature. These are so-called monolithic adsorbents consisting of a three-dimensional porous solid or support based on micro-porous membranes of various polymers. Two-dimensional adsorbents with the pores passing from one side to the other are described as adsorption membranes. According to the invention, target substance(s) and/or contaminant(s) are described as “adsorband” and used in the singular, although they can also consist of several different substances. The “capacity” of an adsorbent is understood to mean a quantitative measure for its uptake capacity for adsorband. The capacity is based on a defined quantity of adsorbent.
The present invention concerns non-particulate ion exchange adsorbers. Some examples are mentioned below. In the state of the art, various non-particulate anion and cation exchangers are known. As examples, strong anion exchangers based on adsorption membranes such as Sartobind® Q from Sartorius Stedim Biotech GmbH, Mustang® Q from Pall Corp., Q Membrane from Natrix Separations or monoliths such as CIM® QA from BIA Separations are mentioned. Other examples are weak anion exchangers such as Sartobind® D from Sartorius Stedim Biotech GmbH, Chromasorb® from Millipore or CIM® EDA from BIA Separations. Furthermore, negatively charged adsorption membranes, such as the strong cation exchanger Sartobind® S or the weak cation exchanger Sartobind® C from Sartorius Stedim Biotech GmbH, strong cation exchange membranes Mustang® S from Pall Corp., S Membrane from Natrix Separations or strong cation exchangers based on monoliths such as for example CIM® SO3 or weak cation exchangers based on monoliths such as for example CIM® CM from BIA Separations are known in the state of the art.
The capacity of an ion exchanger is understood to mean a quantitative measure for its uptake capacity for exchangeable counter-ions. A distinction must be made between the total capacity and the usable capacity. While the total capacity states the total quantity of exchangeable counter-ions, the usable capacity relates only to that fraction which can be utilized under the particular operating conditions (e.g. pH of the solution, concentration of the solution, nature of the counter-ions). Adsorbands can be single molecules, associations or particles, which are preferably proteins or other substances of biological origin. Target substances can for example be recombinant proteins, such as for example monoclonal antibodies. Contaminants can for example be viruses, proteins, amino acids, nucleic acids, endotoxins, protein aggregates, ligands or parts thereof. The removal of contaminants the absence whereof is necessary or desirable for technical, regulatory or other reasons is described as “negative adsorption”.
Most contaminant removal applications are at present operated with conventional chromatography gels. These are particulate in form and are operated in the form of packings in columns. After filling of the column with the medium, a test for function and integrity follows. For this, the theoretical plate number/HETP and the asymmetry of the column packing are determined with suitable solutions of non-binding molecules such as acetone or cooking salt. On the basis of reference samples, the quality of the column packing and suitability for the chromatography step can be determined. The chromatography columns are markedly overdimensioned in order to achieve adequate flow rates. The columns are reused, which signifies considerable cleaning and validation expenditure.
The implementation of chromatographic separations by means of adsorption membranes is also called membrane chromatography. The term adsorption membrane should be understood as a general term for various types of adsorption membranes, such as ion exchange membranes, affinity membranes, hydrophobic membranes or activated membranes. Since filtration effects are most likely undesired, the pore sizes of the adsorptive membranes used on the industrial scale mostly lie in the range of >0.4 μm. In contrast to particulate adsorbents, adsorption membranes offer the possibility of forcing medium volume flow by application of a hydraulic pressure difference between the two sides of their surface, whereby instead of purely diffusive transport of the adsorband in the direction of a concentration gradient into the inside of the adsorbent, convective material transport is attained, which can take place very much faster with high volume flow rate. Thereby a disadvantage inherent to the particulate adsorbents, which is described as “diffusion limitation”, which consists in that with increasing adsorband particle size and increasing adsorband molecular mass the time necessary for establishment of the adsorption equilibrium increases considerably, which results in a worsening of the kinetics, can be avoided. Because of the described advantages of adsorption membranes, these are preferably used in processes wherein the adsorband is present in the medium in very low concentration relative to the capacity of the matrix, so that a large volume of the medium can be processed per unit area of the adsorbent before exhaustion of its capacity.
Typical applications are in the field of negative adsorption, e.g. the removal of contaminants such as DNA, viruses, host cell proteins (HCP), CHOP (Chinese hamster ovary proteins) and/or endotoxins from antibody-containing solutions with positively charged adsorption membranes. This can (may) proceed irreversibly if the adsorbent is to be used only once. The breakthrough of contaminants is a critical factor in validated biopharmaceutical processes. The host cell proteins represent a broad spectrum of different cell proteins with different isolectric points (pI) and different size and affinity to the adsorbent. The concentration and composition of the contaminants depend on the expression system and on the upstream purification steps. Typical concentrations of host cell proteins in a protein A pool lie in the range 500-5000 ppm (ng/mg antibody) and in the range 50-500 ppm after a further CEX step (cation exchange step). The virus depletion is stated as the LRV (log reduction value). It corresponds to the negative base ten logarithm of the ratio of the virus concentration in the starting medium to the virus concentration in the filtrate. Hence an LRV of 5 means that 99.999% of the viruses have been removed by the adsorber. Similarly, the depletion of endotoxins is stated as the LRV.
Adsorption membranes are in general used in modules/capsules which are also described as “membrane adsorbers”. They consist of a housing in which mostly one or preferably several layers of an adsorption membrane are installed. The adsorption membrane is sealed in the housing such that the flow is obligatorily through the membrane layers. The types resemble the modules customary in membrane filtration (e.g. wound module, stack module, etc.). The adsorber is as a rule supplied ready for connection, hence packing of the adsorber by the user is no longer necessary. The design and the shape of membrane adsorbers is adapted to the rapid mode of operation compared to the particulate chromatography columns. In the case of membrane adsorbers, the ratio of adsorption membrane stack height to incident flow area is orders of magnitude smaller than with chromatography columns. The quantities of adsorption membranes needed are as a rule markedly below those of chromatography gels. As a result, the influence of the dead volumes and the adsorber periphery (tubes, pipes, connections, detectors) is also greater than with conventional chromatography columns. The validation methods used for chromatography, such as the determination of the plate number/HETP or the asymmetry of the column packing, are thus rather insensitive and only usable for membrane adsorbers to a very limited extent.
The following criteria should be fulfilled and documented in the validation of an adsorbent installed in the process so that operation appropriate for the application is ensured and the regulatory requirements are fulfilled:    A. Are the correct functional groups present?    B. Is a sufficient quantity of functional groups present?    C. Is a sufficient quantity of functional groups attained during operation of the adsorbent?    D. Is the membrane structure, the membrane stack and the attachment of the membrane to the housing fault-free?
If all these four criteria are fulfilled for an adsorber, according to the invention the integrity of this adsorber is established.
Central to the validation of membrane adsorber systems by the manufacturer are measurements of different parameters, such as for example volume flow rate, binding capacity for model molecules, ligand density, mechanical stability, chemical compatibility and extractable substances. Analogously to the columns, the corresponding tests for functionality and integrity must also be conducted with the membrane adsorbers.
One of the methods used is an integrity test by means of a test device which was developed for sterile-filtering flat filters and filter candles. An example of a commercially available device is the Sartocheck® 4 from Sartorius Stedim Biotech GmbH. Here, the diffusion of air through a membrane stack wetted with water is determined and compared with an intact reference membrane stack. If the diffusion is above a predefined reference value, then a defect is present in the membrane stack. However, this method only yields information about the point D stated above and hence is only valid to a limited extent.
In one method (U.S. Pat. No. 7,281,410 B2, Oct. 16, 2007, Phillips, “Method for determining an effective Peclet number for a membrane device” and US Patent Application Publication US 2003/0089664 A1, May 15, 2003, Phillips, “Membrane Adsorber Device”), the determination of the Peclet number of a membrane adsorber is effected by the steps a) equilibration of the membrane adsorber with an equilibration buffer, b) loading of the membrane adsorber with a known concentration of a specific adsorband in an equilibration buffer, c) detection of the breakthrough of the adsorband as a function of time, loading volume and other suitable variables which are linked with the quantity of the adsorband loaded, d) analysis of the breakthrough curve in order to determine the relevant flow characteristics of the membrane adsorber by calculation of the sharpness of the breakthrough curve, and e) comparison of the results from step d) with a known intact membrane adsorber in order to determine the effective Peclet number. As the adsorband, for example tosylglutamic acid is used, the breakthrough whereof is detected by detection of the UV absorption.
A further method (US Patent Application Publication US 2008/0299672 A1, Dec. 4, 2008, Nochumson et al., “System and method for testing chromatography media and devices”) describes a method for the determination of the integrity of a chromatography membrane welded into a housing, wherein the membrane is subjected to pulsed application of an adsorband e.g. adenosine monophosphate (AMP) under standard conditions, then the bound AMP is eluted with buffer solution and the concentration of the AMP in the eluate as a function of time is measured by UV absorption at 260 nm. The extinction coefficient-time curve thus obtained is compared with the extinction coefficient-time curve of an intact reference module. On occurrence of a defect (hole), in contrast to the intact reference module, early UV absorption occurs.
Both methods known in the state of the art use a “non-process” organic adsorband which is first adsorbed onto the adsorbent in a suitable buffer. In the method according to US 2008/0299672 A1, the adsorband must be eluted from the adsorbent. This represents a major and decisive disadvantage, since it must always be shown that the adsorband has been fully removed from the adsorbent and from the process medium or product. In some cases, the adsorband must be removed in a downstream process step. For regulatory, economic and process safety reasons, this represents a significant limitation. Further, the methods exhibit a relatively low sensitivity of detection via UV absorption and hence exhibit relatively low precision.
For ion exchangers, a pH titration curve (pH of the solution as a function of the quantity of alkali solution or acid added) give information about the number and the pK of the active groups. In a direct pH titration, a defined quantity of ion exchanger with or without addition of salt is titrated directly with equilibrated alkali solution or acid and the pH of the solution is measured after each addition. After each addition of alkali solution or acid, establishment of the equilibrium between ion exchanger and solution must be awaited. This can take a few hours to weeks. In Journal of Chromatography A, 1065 (2005) 29-38, a method for the determination of the quantity of ion exchange groups on a chromatography support is described. After saturation of the adsorbent with a concentrated buffer solution, a low concentration buffer solution at the same pH is passed through the adsorbent. It could be shown that the change in pH with time is a measure of the number of charged groups on the adsorbent. However, the pH change lies in the range of max. 1 pH step and hence this method is insensitive for the detection of faults in membrane adsorbers in the context of a validation.
The present invention is based on the objective of providing a validation method for non-particulate ion exchange adsorbers which enables highly sensitive, robust, simple, non-destructive testing of their integrity and functionality. Preferably, aids (e.g. measuring instruments, test solutions) which signify no impairment of the function of the adsorbent or the product quality nor require any additional process steps or chemicals should be used for the validation.